I’m working on Staphylococcus aureus for typing this bacteria according to VNTR using six primers for this, one of these primers is specific for spa gene and the expected size is 290bp, but after agarose gel electrophoresis the band size was less than 200bp while the same samples checked by capillary electrophoresis and the band size was 290bp. Any one can explain this please?
Apparent sizes of dna and proteins separated electrophoretically depend greatly on the percentage of agarose or PAG as well as the cross linking in PAG and the shape and charge of the molecule.. In PAGE capillary electrophoresis the sample is run under denaturing conditions so all molecules are te same shape so run according to their size. In agarose gels the samples are not denatured so can have different conformations,different bases are on the outside of the molecule so the different charge on the molecule means they can move at different speeds therefore apparent size. The charge on the molecule also means different amounts of water molecukles stick to the dna so increasing its apparent size. I attach a sheet showing how the same dye molecules can run at very different sizes in various gels
Apparent sizes of dna and proteins separated electrophoretically depend greatly on the percentage of agarose or PAG as well as the cross linking in PAG and the shape and charge of the molecule.. In PAGE capillary electrophoresis the sample is run under denaturing conditions so all molecules are te same shape so run according to their size. In agarose gels the samples are not denatured so can have different conformations,different bases are on the outside of the molecule so the different charge on the molecule means they can move at different speeds therefore apparent size. The charge on the molecule also means different amounts of water molecukles stick to the dna so increasing its apparent size. I attach a sheet showing how the same dye molecules can run at very different sizes in various gels
Hi,
Capillary electrophoresis needs size marker (you can use a DNA ladder, no special marker needed) run side by the samples for the first calibration. Please be sure that your size marker is properly identified to the software (band sizes and amounts). If you calibrate correctly, there should not be that much difference.
Sincerely
In agarose gel electrophoresis of DNA, particularly sizes less than 300 bp, the DNA fragments are smaller than the gel pores, so they differently than larger DNA fragments.
As Paul Rutland noted, agarose gel electrophoresis is not performed in denaturing conditions, so the fragments run based on electrophoretic mobility, not necessarily on size. This is an old finding from protein electrophoresis studies: proteins run very differently on SDS-PAGE vs PAGE without SDS.
If your fragment has secondary structure, such as an inverted repeat, it would run faster than its' true length. This would NOT be the case in PAGE, which is run in denaturing conditions. I suspect that is the basis of your findings.
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